226 G. WALD VOL. 4 (1950) 



and are hence protein-free, they still behave as pn indicators, and are therefore still 

 in the coupled condition. 



Not only do the retinenes form a variety of retinal complexes, but normally they 

 migrate from one such association to another. One such migration is established by the 

 present experiments. Rhodopsin and retinene reductase are different proteins. Retinenes 

 originates on rhodopsin protein, but it must transfer to the reductase protein preparatory 

 to its reduction. RetinenCa is involved in a like situation. Such changes of the molecules 

 with which the retinenes are coupled must play an important part in retinal metabolism. 



SUMMARY 



The retinenej which results from the bleaching of rhodopsin now appears to be vitamin Aj 

 aldehyde. Morton et al. have given the best evidence for this, and have shown that retinene^ can 

 be prepared by the mild oxidation of vitamin A^. A simple procedure is described for performing 

 this process chromatographically on a column of manganese dioxide. 



In the retina, retinencj is converted irreversibly to vitamin Aj^ by an enzyme system in which 

 reduced cozymase (reduced Coenzyme I, DPN-Hj) serves as coenzyme. The essential process is the 

 transfer of two hydrogen atoms from DPN-Hj to retinencj, reducing its aldehyde group to the primary 

 alcohol group of vitamin A^. 



The enzyme system which performs this reduction can be assembled in solution from the fol- 

 lowing components: the coenzyme, DPN-Hjl as substrate, synthetic retinene^; and the apoenzyme 

 extracted with dilute salt solutions from homogenized frog or cattle retinas. The apoenzyme is 

 non-dialysable, is precipitated by half-saturated ammonium sulphate, and is destroyed by heating 

 at 100° C within 30 seconds. Its pn optimum lies at about 6.5. 



In the rods of freshwater fishes, a parallel enzyme system reduces retinenej to vitamin Aj. 

 This can be assembled from the following components, all in true solution: the coenzyme, DPN-Hgl 

 as substrate, synthetic retinenej, prepared by the chromatographic oxidation of vitamin Ag on 

 manganese dioxide; and the apoenzyme extracted with dilute salt solutions from freshwater fish 

 retinas (sunfish, yellow perch). 



The apoenzyme from frog retinas reduces retinene2 as effectively as retinenej. Similarly the 

 fish apoenzyme acts equally well upon both retinenes. One need consider only one apoenzyme, retinene 

 reductase, which together with one coenzyme, DPN-Hj, reduces either of the retinenes to the cor- 

 responding vitamin A. 



The retinene reductase system brings a second vitamin into the chemistry of rod vision. It 

 presents the novel phenomenon of one vitamin regenerating another, for the central component of 

 DPN-H, is nicotinamide, the anti-pellagra factor of the vitamin B complex. 



Rhodopsin solutions and retinal homogenates rapidly lose their power to reduce the retinenes, 

 through destruction of their DPN by a nucleotidase. Rhodopsin solutions which have lost their 

 activity in this way are re-activated by the addition of new DPN-Hj. The coenzyme can also be 

 protected by the presence of free nicotinamide and of a-tocopheryl phosphate. 



On addition to the enzyme system, the synthetic retinenes rapidly couple with other molecules, 

 and primarily with protein. The normal state of the retinenes in retinas and retinal extracts is a labile 

 equilibrium between the free and the coupled conditioft. The retinenes couple with a variety of retinal 

 molecules, and migrate freely from one to the other. 



RfiSUMfi 



Le retinene^, qui resulte du blanchissement de la rhodopsine, apparait maintenant comme 6tant 

 I'aldehyde de la vitamine A^. Morton et collab. en ont donne la meilleure preuve en montrant que 

 le retinenci pent etre prepare par une oxydation menagee de la vitamine Aj. Un proc6d6 simple 

 est decrit, qui permet d'effectuer cette operation par chromatographic sur une colonne de bioxyde 

 de manganese. 



Dans la rdtine, le retinenej est converti irr6versiblement en vitamine Aj par un systeme enzy- 

 matique dans lequel la cozymase I r^duite (DPN-H2) sert de coenzyme. Le processus consiste essen- 

 tiellement en un transfert de deux atomes d'hydrogene du DPN-Hg sur le r^tinenej, r^duisant sa 

 fonction aldehydique en fonction alcoolique primaire de la vitamine Aj. 



Le systeme enzymatique qui cffectue cette r6duction pent etre constitu6 en solution a partir 



References p. 228. 



